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1.
Mol Biol (Mosk) ; 58(2): 295-304, 2024.
Artigo em Russo | MEDLINE | ID: mdl-39355886

RESUMO

Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells that are capable of vasculogenic mimicry that is inhibited upon the action of an inhibitor of vasculogenic mimicry. Here, we show that the drug induces a strong upregulation of 50 genes that control the cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes that control different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude that one way for coordinated regulation of large groups of genes is regulation simultaneously by multiple transcription factors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma , Humanos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/tratamento farmacológico , Linhagem Celular Tumoral , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/biossíntese , Ciclo Celular/efeitos dos fármacos
2.
Vet Sci ; 11(10)2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39453043

RESUMO

Roxarsone (ROX) is widely used as a feed addictive for livestock and poultry. ROX promotes angiogenesis, which can lead to health problems, and it is necessary to identify methods to counter this angiogenic effect of ROX. The VEGF/VEGFR2 signaling pathway is involved in the growth and reconstruction of new blood vessels during angiogenesis. In this study, a recombinant lentiviral vector encoding Vegfr2 shRNA was transfected into rat vascular endothelial cells and used in mouse matrigel plug and melanoma xenograft models to investigate its potential to regulate ROX-induced angiogenesis and tumor growth. Treating endothelial cells with ROX increased cell proliferation, migration, and a tube-like structure of growth relative to the control group. The addition of the lentiviral Vegfr2-silencing vector significantly attenuated the effects of ROX on endothelial cells. The hemoglobin content of mouse matrigel plugs treated with ROX was increased significantly. This effect was dramatically attenuated by the co-administration of shRNA targeting Vegfr2. The volume, weight and CD34 staining of the melanoma xenograft tumors increased by ROX were also attenuated by Vegfr2 silence. These results indicate that the down-regulation of VEGFR2 protein plays an inhibitory role in the ROX-promoted angiogenesis in vivo and in vitro. These data support the targeting of Vegfr2 gene as an effective means to treat ROX-induced angiogenesis and tumor growth.

3.
Front Immunol ; 15: 1451154, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39355235

RESUMO

Introduction: The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Methods: Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). Results: No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. Discussion: This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.


Assuntos
Infecções por Coronavirus , Intestino Delgado , Vírus da Diarreia Epidêmica Suína , Animais , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Intestino Delgado/imunologia , Intestino Delgado/virologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/imunologia , Laminina , Combinação de Medicamentos , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , Suscetibilidade a Doenças , Colágeno/metabolismo , Organoides/virologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Proteoglicanas , Células Cultivadas
4.
Cell Rep ; 43(11): 114874, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39423129

RESUMO

Human neural organoid models have become an important tool for studying neurobiology. However, improving the representativeness of neural cell populations in such organoids remains a major effort. In this work, we compared Matrigel, a commercially available matrix, to a neural cadherin (N-cadherin) peptide-functionalized gelatin methacryloyl hydrogel (termed GelMA-Cad) for culturing cortical neural organoids. We determined that peptide presentation can tune cell fate and diversity in gelatin-based matrices during differentiation. Of particular note, cortical organoids cultured in GelMA-Cad hydrogels mapped more closely to human fetal populations and produced neurons with more spontaneous excitatory postsynaptic currents relative to Matrigel. These results provide compelling evidence that matrix-tethered signaling peptides can influence neural organoid differentiation, opening an avenue to control stem cell fate. Moreover, outcomes from this work showcase the technical utility of GelMA-Cad as a simple and defined hydrogel alternative to Matrigel for neural organoid culture.

5.
Int J Mol Sci ; 25(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39273240

RESUMO

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína 1 Inibidora de Diferenciação , Proteína 2 Inibidora de Diferenciação , Proteínas Inibidoras de Diferenciação , Melanoma , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Linhagem Celular Tumoral , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fenótipo , Diferenciação Celular/genética , Epigênese Genética , Combinação de Medicamentos , Colágeno , Proteoglicanas , Laminina , Proteínas de Neoplasias
6.
J Leukoc Biol ; 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39283955

RESUMO

Intravenously administered perfluorocarbon nanoemulsion (PFC) are taken up by phagocytic immune cells which enables the non-invasive visualization of inflammatory hot spots by combined 1H/19F magnetic resonance imaging (MRI). However, little is known about the influence of inflammatory stimuli on cellular uptake and biodistribution of PFCs. Here, we systematically investigated the impact of inflammation induced by subcutaneous implantation of Matrigel/lipopolysaccharide (Matrigel/LPS) or myocardial infarction (MI; 50 minutes ischemia reperfusion) on PFC-uptake and biodistribution in C57BL/6J mice. We detected strong 19F signals in Matrigel/LPS plugs and infarcted hearts, which were completely absent in controls. Cellular uptake of PFCs was increased in neutrophils isolated from the blood and Matrigel/LPS plugs, whereas uptake by monocytes was only slightly elevated. In contrast, MI caused only a moderate early increase of PFC-uptake in monocytes and neutrophils. Interestingly, the inflammatory model did also affect the biodistribution of the PFCs. The blood half-life of PFCs was slightly increased after Matrigel/LPS implantation, whereas it was reduced after MI. Compared to controls, the 19F signal of the liver was significantly stronger in Matrigel/LPS, but not in MI animals. Interestingly, stimulation of primary immune cells and RAW264.7 macrophages with LPS had no effect on PFC-uptake, whereas CRP-incubation elevated internalization of PFCs at least in RAW264.7 cells. In conclusion, we show that the cellular PFC-uptake can differ between individual inflammatory conditions. This is an important aspect that has to be considered for the proper interpretation of 1H/19F MRI data obtained from inflammatory hot spots.

7.
Biomed Phys Eng Express ; 10(6)2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39178888

RESUMO

The absence of effective extracellular matrix to mimic the natural tumor microenvironment remains a significant obstacle in cancer research. Matrigel, abundant in various biological matrix components, is limited in its application due to its high cost. This has prompted researchers to explore alternative matrix substitutes. Here, we have investigated the effects of the extracellular matrix derived from pig small intestinal submucosa (ECM-SIS) in xenograft tumor modeling. Our results showed that the pig-derived ECM-SIS effectively promotes the establishment of xenograft tumor models, with a tumor formation rate comparable to that of Matrigel. Furthermore, we showed that the pig-derived ECM-SIS exhibited lower immune rejection and fewer infiltrating macrophages than Matrigel. Gene sequencing analysis demonstrated only a 0.5% difference in genes between pig-derived ECM-SIS and Matrigel during the process of tumor tissue formation. These differentially expressed genes primarily participate in cellular processes, biological regulation, and metabolic processes. These findings emphasize the potential of pig-derived ECM-SIS as a cost-effective option for tumor modeling in cancer research.


Assuntos
Matriz Extracelular , Laminina , Animais , Matriz Extracelular/metabolismo , Suínos , Camundongos , Humanos , Proteoglicanas , Colágeno/química , Microambiente Tumoral , Mucosa Intestinal/metabolismo , Combinação de Medicamentos , Linhagem Celular Tumoral , Intestino Delgado , Géis , Neoplasias
8.
Exp Cell Res ; 442(2): 114188, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39128553

RESUMO

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

9.
Bio Protoc ; 14(15): e5040, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39131196

RESUMO

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

10.
Front Immunol ; 15: 1401751, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39119341

RESUMO

Introduction: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. Methods: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. Results: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1ß treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Discussion: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.


Assuntos
Matriz Extracelular , Homeostase , Laminina , Neuroglia , Animais , Matriz Extracelular/metabolismo , Neuroglia/metabolismo , Neuroglia/imunologia , Camundongos , Laminina/metabolismo , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/imunologia , Células Cultivadas , Combinação de Medicamentos , Colágeno/metabolismo , Camundongos Endogâmicos C57BL , Proteoglicanas/metabolismo
11.
Adv Biol (Weinh) ; 8(9): e2400184, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38971965

RESUMO

Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.


Assuntos
Sobrevivência Celular , Matriz Extracelular , Ácido Hialurônico , Hidrogéis , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/química , Linhagem Celular Tumoral , Feminino , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Encéfalo/patologia , Encéfalo/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Alicerces Teciduais/química , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Poliésteres
12.
Int J Nanomedicine ; 19: 6201-6228, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38911499

RESUMO

Due to their ability to replicate the in vivo microenvironment through cell interaction and induce cells to stimulate cell function, three-dimensional cell culture models can overcome the limitations of two-dimensional models. Organoids are 3D models that demonstrate the ability to replicate the natural structure of an organ. In most organoid tissue cultures, matrigel made of a mouse tumor extracellular matrix protein mixture is an essential ingredient. However, its tumor-derived origin, batch-to-batch variation, high cost, and safety concerns have limited the usefulness of organoid drug development and regenerative medicine. Its clinical application has also been hindered by the fact that organoid generation is dependent on the use of poorly defined matrices. Therefore, matrix optimization is a crucial step in developing organoid culture that introduces alternatives as different materials. Recently, a variety of substitute materials has reportedly replaced matrigel. The purpose of this study is to review the significance of the latest advances in materials for cell culture applications and how they enhance build network systems by generating proper cell behavior. Excellence in cell behavior is evaluated from their cell characteristics, cell proliferation, cell differentiation, and even gene expression. As a result, graphene oxide as a matrix optimization demonstrated high potency in developing organoid models. Graphene oxide can promote good cell behavior and is well known for having good biocompatibility. Hence, advances in matrix optimization of graphene oxide provide opportunities for the future development of advanced organoid models.


Assuntos
Grafite , Organoides , Organoides/efeitos dos fármacos , Organoides/citologia , Animais , Grafite/química , Grafite/farmacologia , Humanos , Proliferação de Células/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Combinação de Medicamentos , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células em Três Dimensões/métodos , Camundongos , Laminina/química , Laminina/farmacologia , Colágeno , Proteoglicanas
13.
ACS Appl Mater Interfaces ; 16(25): 31922-31935, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38874539

RESUMO

Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.


Assuntos
Diferenciação Celular , Proliferação de Células , Colágeno , Combinação de Medicamentos , Laminina , Células-Tronco Neurais , Polilisina , Proteoglicanas , Polilisina/química , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Laminina/química , Laminina/farmacologia , Colágeno/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteoglicanas/química , Proteoglicanas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Camundongos
14.
Mater Today Bio ; 26: 101065, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38706731

RESUMO

The recent FDA decision to eliminate animal testing requirements emphasises the role of cell models, such as spheroids, as regulatory test alternatives for investigations of cellular behaviour, drug responses, and disease modelling. The influence of environment on spheroid formation are incompletely understood, leading to uncertainty in matrix selection for scaffold-based 3D culture. This study uses atomic force microscopy-based techniques to quantify cell adhesion to Matrigel and cellulose nanofibrils (CNF), and cell-cell adhesion forces, and their role in spheroid formation of hepatocellular carcinoma (HepG2) and induced pluripotent stem cells (iPS(IMR90)-4). Results showed different cell behaviour in CNF and Matrigel cultures. Both cell lines formed compact spheroids in CNF but loose cell aggregates in Matrigel. Interestingly, the type of cell adhesion protein, and not the bond strength, appeared to be a key factor in the formation of compact spheroids. The gene expression of E- and N-cadherins, proteins on cell membrane responsible for cell-cell interactions, was increased in CNF culture, leading to formation of compact spheroids while Matrigel culture induced integrin-laminin binding and downregulated E-cadherin expression, resulting in looser cell aggregates. These findings enhance our understanding of cell-biomaterial interactions in 3D cultures and offer insights for improved 3D cell models, culture biomaterials, and applications in drug research.

15.
BMC Biotechnol ; 24(1): 36, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38796454

RESUMO

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.


Assuntos
Colágeno , Combinação de Medicamentos , Laminina , Células-Tronco Mesenquimais , Proteoglicanas , Ratos Sprague-Dawley , Alicerces Teciduais , Cicatrização , Animais , Laminina/química , Proteoglicanas/química , Colágeno/química , Humanos , Ratos , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais/química , Diferenciação Celular , Proliferação de Células , Gengiva/citologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Engenharia Tecidual/métodos , Masculino , Mucosa Bucal/citologia
16.
Methods Mol Biol ; 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38797794

RESUMO

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

17.
Cancer Cell Int ; 24(1): 180, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38783299

RESUMO

BACKGROUND: Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS: Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS: UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS: Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

18.
ACS Biomater Sci Eng ; 10(5): 3218-3231, 2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38593429

RESUMO

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.


Assuntos
Diferenciação Celular , Matriz Extracelular Descelularizada , Hidrogéis , Células-Tronco Pluripotentes Induzidas , Organoides , Placenta , Medula Espinal , Humanos , Organoides/citologia , Organoides/metabolismo , Organoides/efeitos dos fármacos , Feminino , Placenta/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Gravidez , Hidrogéis/química , Hidrogéis/farmacologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Diferenciação Celular/efeitos dos fármacos , Matriz Extracelular Descelularizada/farmacologia , Matriz Extracelular Descelularizada/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Laminina/farmacologia , Laminina/química
19.
Biomater Adv ; 160: 213847, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38657288

RESUMO

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.


Assuntos
Técnicas de Cultura de Células em Três Dimensões , Colágeno , Combinação de Medicamentos , Células Epiteliais , Hidrogéis , Laminina , Peptídeos , Proteoglicanas , Laminina/farmacologia , Laminina/química , Hidrogéis/química , Hidrogéis/farmacologia , Proteoglicanas/farmacologia , Proteoglicanas/química , Colágeno/química , Colágeno/farmacologia , Peptídeos/farmacologia , Peptídeos/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/citologia , Humanos , Feminino , Técnicas de Cultura de Células em Três Dimensões/métodos , Sobrevivência Celular/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Glândulas Mamárias Humanas/citologia , Organoides/efeitos dos fármacos , Organoides/citologia , Técnicas de Cultura de Células/métodos
20.
bioRxiv ; 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38585922

RESUMO

Background: Primary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing ß-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.

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